The present invention relates to an apparatus and method for growing anaerobic microorganisms. The apparatus is comprised of a specially designed culture dish which can be reconfigured such as by inverting the dish to produce an anaerobic environment. An oxygen reducing agent such as a biocatalytic oxygen reducing agent can also be incorporated into the media present in the apparatus together, in some circumstances, with a substrate. The biocatalytic oxygen reducing agent and the substrate present in the media react with oxygen enclosed in the culture dish to create an environment suitable for growing and maintaining anaerobic microorganisms.
Microorganisms are important to our well being. This is evident in healthcare, agriculture and industry. To be able to simply and quickly isolate and grow microbes is economically important. For example, being able to quickly and specifically isolate and identify a microbe responsible for infection is important in human health care field. This basic technique is also important in the agriculture industry. Large scale processing of food requires constant microbial monitoring. The speed and efficiency at which this can be done determines the length of time finished food products must be held in storage before they can be distributed for sale.
Control of the environment is necessary for control of microbial growth. In particular, control of oxygen content in the immediate environment is crucial for microbial growth. Microorganisms can be divided into groups based on their need for, and tolerance of, oxygen. There are those that require oxygen to grow. These are "aerobes". Some microorganisms are able to grow with or without oxygen. These are "facultative anaerobes". Another group of microorganisms can grow only in the presence of very low levels of oxygen. These are the "microaerophiles". Finally, some microorganisms can not tolerate oxygen. They are inhibited by it or may be killed by it. These are the "anaerobes".
This fundamental property of microorganisms, that is their ability to grow in or tolerate oxygen, is used daily to isolate, grow, and manipulate them. One basic technique in microbiology, is the plating method. This generally involves the use of a dish, developed by Petri (i.e. "Petri dish") in 1880's, and solidified (agar or gelatin-based) medium.
A Petri dish is usually a round, shallow, flat-bottomed, glass or plastic dish (often e.g. 10 cm diameter) with a vertical side, that cooperates with a similar, slightly larger structure which forms a loosely-fitting lid. Petri dishes are used in microbiology, e.g., for the preparation of plates.
The purpose of the Petri dish is to provide a controlled environment for selectively growing microbes. The dish is sterilized and designed to maintain a sterile environment inside while freely exchanging gases, normally air, with the outside environment.
The medium utilized in conjunction with the Petri dish can be formulated to provide a necessary and selective environment for a specific microorganism. Solid medium in a Petri dish can be prepared using aseptic technique by pouring sterile molten or liquid (agar- or gelatin-based) medium into a Petri dish to a depth of 3-5 mm and allowing it to set. Generally, freshly poured plates to be used for separation and/or generation of microbes should be left for 30 minutes in a 45.degree. C. hot-air incubator with the lid partly off so that the surface moisture can evaporate. Such "drying" before inoculation prevents unwanted spreading of the inoculum in the surface film of the moisture.
The solid medium surface inside the dish provides a place to grow microorganisms. By inoculating (or "plating") the surface of the agar in a controlled way (i.e. "streaking") , single colonies of a microorganism can be obtained. With this technique the microbiologist can separate microbes one from another. Isolation and purification is mandatory to further characterization and study. Using this dish design, a microbiologist can isolate and grow the great majority of microorganisms known today.
Working with microbes that are microaerophiles or anaerobes poses a problem. The culture dishes for these microbes must be incubated in a controlled gaseous environment that lacks oxygen, or at least most of the oxygen, found in air. This is done by placing the culture or Petri dish containing medium inside a container that is sealed from the outside atmosphere. For one or a few dishes, a sealable bag or jar (i.e., "Brewer Jar") is used (Becton Dickinson Microbiology Systems, 1994 Catalog, p 89-p 94). In this case, chemicals and a catalyst (see U.S. Pat. No. 4,287,306 issued Sep. 1, 1982 to Brewer entitled "Apparatus for Generation of Anaerobic Atmosphere") are placed inside the container that, when activated chemically, reacts with the oxygen in the container, thus removing it. The catalyst is necessary to bring about the reaction at low temperatures in a short time.
In addition, for many culture dishes, a sealed table-top chamber can be used (Anaerobe Systems, San Jose, Calif.). This chamber is evacuated and flushed with inert gases, such as nitrogen and/or carbon dioxide. Sometimes chemicals and a catalyst are used to consume the oxygen inside the chamber and fresh, inert gas is supplied as needed. The microbiologist works with the culture dishes inside of this chamber through ports fitted with gloves. A means is provided for introducing materials into and taking items out of the chamber without breaching the anaerobic environment inside.
Work with microaerophiles and anaerobes under these conditions is labor intensive, difficult, expensive, and time consuming. The microbiologist is often frustrated by having to wait for the slowest growing microbe in order to retrieve all culture dishes from a bag or jar since once the bag or jar is opened, the microbes are exposed to oxygen. A failure in the system can be catastrophic for all of the microbial isolates inside.
To overcome many of these problems (see U.S. Pat. No. 2,348,448 issued May 9, 1944 to Brewer entitled "Apparatus for the Cultivation of Anaerobic and Microaerophilic Organisms") Brewer developed a culture dish lid (i.e., "Brewer Lid") that formed a seal between a ring inside the lid with the agar or gelatin-based surface. Within the dish, a very small, defined headspace is formed by the lid and the agar surface. An anaerobic environment is created inside this trapped headspace by reacting oxygen with chemical reducing agents, such as thioglycollate, incorporated in the medium. The limited volume of the headspace is important to the function of the Brewer Lid.
However, a number of drawbacks exist in the use of the Brewer Lid. The capacity and the rate for oxygen removal is limited by the sensitivity of the microorganism to the chemical reducing agent in the medium (see "Mechanism of Growth Inhibitory Effect of cysteine on Escherichia coli." of Kari, et al., J. Gen. Microbiol., 68, 1971, p. 349 and "Methods for General and Molecular Bacteriology", Editor: Gerherdt, American Society for Microbiology, 1994, p. 146.). Moreover, the lid is made of heavy glass and is expensive. It is available today (Kimble Glass Company, Vineland, N.J.), but is not widely used because of serious limitations that include cost, handling difficulties, and poor response of anaerobic microorganisms.
Another limitation is caused by the material of construction. The glass Brewer Lid is made very heavy to insure a good seal between the ring inside the Brewer Lid and the agar surface. Cultures dish bottoms fitted with the heavy Brewer Lid are not easy to handle or to move about. They can not be stacked inside an incubator. Thus, precious incubator space is wasted. Stacked dishes crush the agar medium of the lowest dishes in the stack, because of the weight of the dishes above them. This causes the headspace above the agar to collapse resulting in contact between the inside of the Brewer Lid and the agar surface. When this happens, the microbial growth on the surface is spread out and separation of individual colonies is lost. Motile microbes will migrate and further frustrate separation.
Because of their weight and material of construction, Brewer Lids do not lend themselves to commercial production of pre-made agar or gelatin-based plates. The commercial process requires assembly line filling of the dishes, packaging the filled dishes in stacks, and handling and storing these dishes. Pre-made agar plates are widely used in clinical microbiological laboratories. This limitation of the Brewer Lid is economically significant.
The headspace inside the Brewer Lid formed by the lid and agar surface is very small. This limited headspace is determined by the ability of the chemical reducing agent (H.sub.2 S, cysteine, thioglycollate, etc.) to reduce oxygen in the headspace. The amount of chemical reducing agent used in the medium in turn is constrained by anaerobic microorganism's sensitivity to it. The sum of these limitations is a very small head space that imparts severe problems to the function of the Brewer Lid for its intended purpose, i.e. to grow anaerobic and microaerophilic microorganisms.
Another limitation of the Brewer Lid is that the very limited head space can not hold much moisture. Fresh agar medium is generally greater than 98 percent water. Upon incubation, water in the medium evaporates and condenses upon the upper surface of the inside of the lid. This condensate can become sufficient to fall to the agar surface and to flood it. Under such conditions, the plate is ruined and can not be used for isolation and purification of the microbe.
The very limited headspace imposes still more limitations on the Brewer Lid. No provision is made to incorporate CO.sub.2 into the headspace above the agar surface. This is important for the rapid growth of some microorganisms and may be required by others. Yet this feature should be made optional for the microbiologist, because for some uses of the culture dish the microbiologist may not want to include CO.sub.2 in the headspace. Reports show that CO.sub.2 can change the pH of the medium it contacts. This in turn can interfere with the determination of susceptibility to some antibiotics (see "Effect of CO.sub.2 on Susceptibilities of Anaerobes to Erythromycin, Azithromycin, Clarithromycin, and Roxithromycin", Spangler, et al., Antimicrob. Agents Chemotherapy, 38, p. 20, 1994). Since CO.sub.2 is generated in anaerobic jars and bags by commercial catalysts products, this problem is commonly encountered. CO.sub.2 is a component of the gas used to flush anaerobic chambers and incubators too.
Another desired feature for a self contained culture dish is an indicator to show that the headspace is anaerobic. These features are difficult to impossible to include in the Brewer Lid because of the very small space between inside the lid top and the agar surface.
Several attempts have been made to design a culture dish that provides a self-contained environment for growing anaerobic microorganisms (see U.S. Pat. No. 2,701,229 issued Feb. 1, 1955 to Scherr entitled "Apparatus for the Cultivation of Microorganisms"; U.S. Pat. No. 3,165,450 issued Jan. 12, 1965 to Scheidt entitled "Anaerobic Culturing Device"; U.S. Pat. No. 4,294,924 issued Oct. 13, 1981 to Pepicelli, et al. entitled "Method and Container for Growth of Anaerobic Microorganisms"; U.S. Pat. No. 4,299,921 issued Nov. 10, 1981 to Youssef entitled "Prolonged Incubation Microbiological Apparatus and Filter Gaskets Thereof"; and U.S. Pat. No. 4,859,586 issued Aug. 8, 1989 to Eisenberg entitled "Device for Cultivating Bacteria") . The fact that the Brewer Lid and none of these inventions are commonly or commercially available or used widely by microbiologists today, attest to their limitations and shortcomings. The need to simplify and reduce the cost for isolating and growing anaerobic and microaerophilic microorganisms still exists today.
It is therefore an object of the present invention to provide an improved apparatus and method for cultivating and/or enumerating anaerobic microorganisms which obviate the above-mentioned disadvantages of the prior art.
Another object of the present invention is to provide an improved anaerobic culturing apparatus which is extremely simple, inexpensive and easy to use and wherein the proper anaerobic environment is produced and maintained in an extremely efficient manner.
These and other additional objects and advantages of the present invention will become apparent from the following description of the invention.